Tau protein production accelerator, and therapeutic or preventive agent and therapeutic or preventive food composition for diseases caused by tau protein deficiency

ABSTRACT

An object of the present invention is to provide: a Tau protein production accelerator containing a natural product as an active ingredient; a therapeutic or preventive agent for a disease caused by Tau protein deficiency; and a therapeutic or preventive food composition for a disease caused by Tau protein deficiency. Provided are: a Tau protein production accelerator containing a dry powder, ground product and/or extract of an earthworm as an active ingredient; a therapeutic or preventive agent for a disease caused by Tau protein deficiency; and a therapeutic or preventive food composition for a disease caused by Tau protein deficiency. The disease caused by Tau protein deficiency is preferably Alzheimer&#39;s disease.

TECHNICAL FIELD

The present invention relates to: a Tau protein production accelerator;a therapeutic or preventive agent for a disease caused by Tau proteindeficiency; and a food composition for the treatment or prevention of adisease caused by Tau protein deficiency.

BACKGROUND ART

Tau proteins are a type of microtubule-associated protein. Tau proteinsare particularly abundant in neurons of the central nervous system andhave a function of binding to a protein called tubulin which mainlyconstitutes a cytoskeleton component, microtubules, and therebystabilizing microtubules and promoting tubulin assembly intomicrotubules. In addition to tubulin, Tau proteins are also known toassociate with other signaling molecules (Src family, PI3K, Fyn) and topromote neurite outgrowth and elongation of nerve growth cones (see, forexample, Non-patent Documents 1 to 3).

Phosphorylation of Tau proteins can occur excessively in vivo. When Tauproteins are excessively phosphorylated, their binding with tubulin isinhibited, as a result of which microtubules are reduced or microtubulesare destabilized and the intracellular substance transport is suppressed(see, for example, Non-patent Documents 4 and 5). Excessivelyphosphorylated Tau proteins aggregate with each other to form aggregatescalled neurofibrillary tangles. Alzheimer's disease and progressivesupranuclear palsy that involve such formation of neurofibrillarytangles are classified into neurodegenerative diseases called tauopathy.

There have been proposed methods of treating a tauopathy by compensatingthe functions of Tau proteins. For example, U.S. Pat. No. 5,580,898suggests the use of paclitaxel [TAXOL (registered trademark)] for thetreatment of Alzheimer's disease patients through stabilization ofmicrotubules. Further, Patent Document 1 describes an effectivetherapeutic method of tauopathy using a microtubule stabilizer,epothilone D.

Meanwhile, mainly in the Oriental countries, earthworm extracts and dryearthworm powders have been used since ancient times as preventiveagents and therapeutic agents for various diseases, and examples oftheir use that have been known include bladder stone-reducing agents,bladder stone excretion-promoting agents, therapeutic agents forjaundice, oxytocics, tonic agents, hair growth agents, aphrodisiacs,antipyretics, therapeutic agents for convulsion, blood circulationpromoters, therapeutic agents for hemiplegia, indirect analgesics,diuretics, antiasthmatics and antihypertensive agents.

However, no report has been made on the use of earthworms for theprevention and treatment of tauopathy such as Alzheimer's disease.

RELATED ART DOCUMENTS Patent Document

Patent Document 1: Japanese Unexamined Patent Application Publication(Translation of PCT Application) No. 2011-522782

Non Patent Documents

Non-patent Document 1: Lee, G. Biochimica et Biophysics Acta (2005)1739: 323-330

Non-patent Document 2: Reynolds, C H et al., THE JOURNAL OF BIOLOGICALCHEMISTRY (2008) 283: 18177-18186

Non-patent Document 3: Nemoto, T et al., Neurochemistry International(2011) 59: 880-888

Non-patent Document 4: Bramblett G T, et al., Neuron (1993) 10:1089-1099

Non-patent Document 5: Yoshida H, et al., J Neurochem (1993) 61: 1183-86

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The treatment of tauopathy such as Alzheimer's disease is believed toinvolve drug administration over a long time; therefore, a drug that issafe and has little side effects is particularly necessary; and there isthus a demand for a naturally derived preventive or therapeutic agentwhich compensates the functions of Tau protein not normally functioningdue to phosphorylation.

In view of the above, an object of the present invention is to provide:a Tau protein production accelerator comprising a natural product as anactive ingredient; a preventive or therapeutic agent for diseases causedby Tau protein deficiency; as well as a food composition andpharmaceutical composition for an improvement of the symptoms ofdiseases caused by Tau protein deficiency.

Means for Solving the Problems

That is, the Tau protein production accelerator according to the presentinvention is characterized by comprising a dry powder, ground productand/or extract of an earthworm as an active ingredient.

The therapeutic or preventive agent for a disease caused by Tau proteindeficiency according to the present invention is characterized bycomprising the above-described Tau protein production accelerator.

The therapeutic or preventive food composition for a disease caused byTau protein deficiency according to the present invention ischaracterized by comprising the above-described Tau protein productionaccelerator.

In the therapeutic or preventive agent for a disease caused by Tauprotein deficiency according to the present invention, it is preferredthat the disease caused by Tau protein deficiency be Alzheimer'sdisease.

In the therapeutic or preventive food composition for a disease causedby Tau protein deficiency according to the present invention, it ispreferred that the disease caused by Tau protein deficiency beAlzheimer's disease.

The method of producing a Tau protein production accelerator accordingto the present invention is characterized by comprising the use of a drypowder, ground product or extract of an earthworm.

The method of producing a therapeutic or preventive agent for a diseasecaused by Tau protein deficiency according to the present invention ischaracterized by comprising the use of a dry-powder, ground product orextract of an earthworm.

The method of producing a therapeutic or preventive food composition fora disease caused by Tau protein deficiency according to the presentinvention is characterized by comprising the use of a dry powder, groundproduct or extract of an earthworm.

The method of promoting Tau protein production according to the presentinvention is characterized by comprising the use of a dry powder, groundproduct and/or extract of an earthworm.

The dry powder, ground product or extract of an earthworm according tothe present invention is for the use in the treatment of a diseasecaused by Tau protein deficiency.

The method of treating or preventing a disease caused by Tau proteindeficiency according to the present invention is characterized bycomprising administration of a dry powder, ground product and or extractof an earthworm to a subject in an effective dose.

EFFECTS OF THE INVENTION

According to the present invention, a Tau protein production acceleratorcomprising a natural product as an active ingredient, a preventive ortherapeutic agent for diseases caused by Tau protein deficiency, as wellas a food composition and pharmaceutical composition for an improvementof the symptoms of diseases caused by Tau protein deficiency can beprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides photographs showing the results of Western blottingwhich was performed to investigate the changes in the amounts of Tauprotein (Tau), Akt, GSK-3β and β-actin produced in rat hippocampalneurons during culture in a dry earthworm powder-dissolved culturemedium (48 hours, 37° C., concentration of dissolved dry earthwormpowder: 100 ng/ml) as well as the changes in the amounts ofphosphorylated Tau protein (Tau), Akt and GSK-3β (pTau, pAkt andpGSK-3β, respectively). The “−” and “+” columns show the results ofculturing rat hippocampal neurons in a culture medium MM5 and a culturemedium prepared by dissolving dry earthworm powder in a culture mediumNM5 (100 ng/ml), respectively. It is noted here that Tau protein, Aktand GSK-3β were evaluated using Ser396, Ser473 and Ser9 as an index ofphosphorylation, respectively.

FIG. 2 provides graphs showing the concentrations of Tau protein (Tau),Akt and GSK-3β as well as the concentrations of phosphorylated Tauprotein (pTau), Akt (pAkt) and GSK-3β (pGSK-3β), which were quantifiedfrom the results of Western blotting shown in FIG. 1 using an imageanalysis software ImageJ64. FIG. 2A shows, from the left, the pTauconcentration and the Tau protein concentration, both of which are basedon β-actin, as well as the ratio of pTau with respect to Tau protein;FIG. 2B shows, from the left, the pAkt concentration and the Aktconcentration, both of which are based on β-actin, as well as the ratioof pAkt with respect to Akt; and FIG. 2C shows, from the left, thepGSK-3β concentration and the GSK-3β concentration, both of which arebased on β-actin, as well as the ratio of pGSK-3β with respect toGSK-3β.

FIG. 3A provides photographs showing the results of Western blottingwhich was performed to investigate the changes in the amounts of Tauprotein (Tau) and β-actin produced in rat hippocampal neurons duringculture In a dry earthworm powder-dissolved culture medium (48 hours,37° C.) in accordance with changes in the concentration of dissolved dryearthworm powder; and FIG. 3B is a graph showing the changes in theconcentration of Tau protein based on β-actin, which were quantifiedusing an image analysis software ImageJ64.

FIG. 4A provides photographs showing the results of Western blottingwhich was performed to investigate the changes in the amounts of Tauprotein (Tau) and β-actin produced in rat hippocampal neurons duringculture in a dry earthworm powder-dissolved culture medium (37°C.,concentration of dissolved dry earthworm powder: 100 ng/ml) inaccordance with changes in the duration of culture period; and FIG. 4Bis a graph showing the changes in the concentration of Tau protein basedon β-actin, which were quantified using an image analysis software ImageJ64.

FIG. 5 shows immunostained fluorescence images of Tau protein (Tau) andpTau in rat hippocampal neurons that were cultured at 37° C. for 48hours in a culture medium NM5 or a culture medium prepared by dissolvingdry earthworm powder in a culture medium NM5 (100 ng/ml). The upper row(None) shows the fluorescence images obtained for the rat hippocampalneurons in the culture medium, while the lower row (RW) shows thefluorescence images obtained tor the rat hippocampal neurons in the dryearthworm powder-dissolved culture medium. The left column shows thefluorescence images of Tau protein; the center column shows thefluorescence images of pTau; and the right column shows the fluorescenceimages of Tau protein and pTau.

MODE FOR CARRYING OUT THE INVENTION

In the method of the present invention, the earthworm used as a rawmaterial is not particularly restricted, and examples of earthworms thatcan be used include Lumbricus rubellus, Lumbricus terrestris, Eiseniafoetida, Allolobophora caliginosa, Dendrobaena octaedra, Allolobophorajaponica Michaelsen, Drawida hattamimizu Hatai, Pherentima divergensMichaelsen, Pherentima communissima, Pheretima agrestis, Pherentimasieboldi Horst, Pheretima hilgendorfi, Pontodrilus matsushimensisIizuka, Tubifex hattai Nomura, and Limnodrilus gotoi Hatai (=L. SocialisStephenson).

In the present invention, the term “dry powder” of an earthworm meanspowder obtained by drying a ground product or extract of an untreated orpretreated earthworm. The term “ground product” of an earth worm meansan untreated or pretreated earthworm ground into a liquid or paste form.The term “extract” of an earthworm means an extract obtained bydissolving an untreated or pretreated earthworm or a ground productthereof in water or an organic solvent and subsequently removing orseparating insoluble fractions. The pretreatment is not particularlyrestricted, and examples thereof include the below-described treatmentfor removal of dirt and the like. Further, the dry powder, groundproduct and extract of an earthworm may also be subjected to apost-treatment, examples of which include granulation, filtration,purification, concentration, dilution and pH adjustment.

The grinding method for obtaining a ground product of an earthworm isnot particularly restricted, and grinding can be performed by using, forexample, a homogenizer, a blender, a homomixer, a grinder or ahigh-pressure cell crushing apparatus.

The extraction method for obtaining an extract of an earthworm is notparticularly restricted and extraction can be performed by, for example,dissolving dry powder or a ground product of the earthworm in anextraction solvent and subsequently removing or separating insolubletractions. Examples of the extraction solvent include water, aqueoussolutions, and organic solvents such as ethanol, acetone and ethylacetate, and these extraction solvents may be used individually, or twoor more thereof may be used in combination. Thereamong, water, ethanolor an aqueous ethanol solution is preferably used.

The drying method for obtaining a dried product of an earthworm is notparticularly restricted, and drying can be performed by a drying methodsuch as freeze-drying, heat-drying or spray-drying. Thereamong,freeze-drying is preferred for the below-described reasons.

In the present invention, the dry powder, ground product or extract ofthe earthworm can be incorporated in an effective amount in accordancewith the purpose thereof. The appropriate amount depends on a variety offactors such as the intended purpose, the route and mode ofadministration and the production method of the dry powder or the likeof the earthworm; however, for the purpose of preventing diseases causedby Tau protein deficiency or treating a mild disease, the appropriateamount is preferably 1 to 15,000 mg/day, more preferably 12 to 1,800mg/day, still more preferably 120 to 180 mg/day, in terms of the weightof the dry powder of the earthworm obtained by removing the digestedmatters remaining in the digestive tract of the earthworm as well as thedirt and the like adhering to the skin of the earthworm as describedbelow, grinding the earthworm and then freeze-drying the resultingground product. Further, for the purpose of treating a severe diseasecaused by Tau protein deficiency, the appropriate amount is preferably 1to 15,000 mg/day, more preferably 18 to 3,600 mg/day, still morepreferably 180 to 360 mg/day.

The forms of the Tau protein production accelerator, therapeutic agent,preventive agent and food composition of the present invention are notparticularly restricted and can be any of a solid form, a powder form, asemisolid form and a liquid form.

In the present invention, the dry powder, ground product or extract ofthe earthworm can be used as is. Alternatively, particularly the Tauprotein production accelerator, therapeutic agent and preventive agentof the present invention may contain a pharmaceutically acceptablecarrier and can be administered orally or parenterally (e.g.,intravenous administration or direct administration to the affectedsite) in the form of a tablet, a granule, a powder, a capsule, a softcapsule, a liquid, an injectable, a suppository or a sustained releaseagent or the like. As the pharmaceutically acceptable carrier, forexample, an excipient, a binding agent, a disintegrant, a fluidizingagent, a lubricant, a coating agent, a suspending agent, a colorant, asweetening agent or a surfactant can be used, and the resultant can bemade into the form of an ordinary pharmaceutical preparation inaccordance with a known method. Further, other therapeutic andpreventive components) and pharmaceutically acceptable additive(s) mayalso be incorporated.

In the present invention, particularly in the Tau protein productionaccelerator and food composition of the present invention, anadditive(s) usually used in food products may also be incorporated.Examples of additives that can be used include an excipient, a bindingagent, a disintegrant, a fluidizing agent, a lubricant, a coating agent,a suspending agent, a colorant, a sweetening agent and a surfactant, andthe resultant can be made into the form of an ordinary food compositionin accordance with a known method. Further, other food products) orfood-derived component(s) may be incorporated as well.

In the present invention, among a dry powder, a ground product and anextract of earthworms, from the standpoint of the storage stability inthe production process, it is preferred to use a dry powder of anearthworm. The dry powder of an earthworm may be dissolved and/ordispersed in a liquid such as water in advance and the resultant may besubsequently mixed with other component(s), examples of which includeconventional carriers and additives that are used pharmaceuticallyand/or in food products.

In the present invention, the disease caused by Tau protein deficiencyis not particularly restricted; however, it is preferably a tauopathy,more preferably one selected from the group consisting of Alzheimer'sdisease. Pick's disease, corticobasal degeneration, progressivesupranuclear palsy, chronic traumatic encephalopathy, frontotemporaldementia and parkinsonism linked to chromosome 17, Parkinsonism-dementiacomplex of Guam, neurofibrillary tangle-predominant senile dementiaaccompanied by neurofibrillary tangles similar to those of amyloidplaque-free Alzheimer's disease, ganglioglioma, gangliocytoma, subacutesclerosing panencephalitis, tuberous sclerosis, Hallervorden-Spatzdisease, frontotemporai dementia, and frontotemporal lobar degeneration.The disease caused by Tau protein deficiency is particularly preferablyAlzheimer's disease.

For oral administration of an earthworm as a raw material, it ispreferred to remove the digested matters remaining in the digestivetract of the earthworm, the dirt adhering to the skin and the like. Inthe present invention, the method for such removal is not particularlyrestricted, and the removal can be performed by a known method. Forexample, a method of allowing a live earthworm to excrete yellow soilcontained in the digestive tract by immersing the earthworm into anaqueous solution of an alkali salt such as a sodium salt or a potassiumsalt (method described in Japanese Unexamined Patent ApplicationPublication Nos. H1-47718, H1-47719, H1-47720 and H1-268639) or a methodof removing castings from the digestive tract of a live earthworm byleaving the earthworm in an aqueous acid solution maintained at 6 to 26°C. for 0.1 to 5 hours (method described in Japanese Unexamined PatentApplication Publication No. H3-72427) can be employed.

In the present invention, as a removal method, it is preferred to bringthe earthworm into contact with the below-described metal chlorideand/or hydroxycarboxylic acid.

The metal chloride is a chloride of at least one metal selected from thegroup consisting of potassium, sodium, magnesium and calcium. That is,the metal chloride is at least one selected from the group consisting ofpotassium chloride, sodium chloride, magnesium chloride and calciumchloride. Further, the metal chloride may also be a mixture of thesemetal chlorides, or a mixture of one or more of these metal chloridesand other harmless components) that can be added to food products.Examples of such mixtures include dietary salts, rock salts and baysalts. The metal chloride can be used by sprinkling it in a powder formover a live earthworm, and this causes a contact between the earthwormand the metal chloride.

After allowing the metal chloride to come into contact with the liveearthworm, it is preferred to bring the live earthworm into contact witha hydroxycarboxylic acid in the below-described manner. Alternatively,the earthworm may be brought into contact with a hydroxycarboxylic acidin the below-described manner without a preceding contact with the metalchloride.

The contact with the hydroxycarboxylic acid can also be made bysprinkling the hydroxycarboxylic acid in a powder form over the liveearthworm. Alternatively, the live earthworm may be immersed in anaqueous solution of the hydroxycarboxylic acid that has a pH of 2 to 5.In cases where the contact with the hydroxycarboxylic acid is made aftera contact with the metal chloride, it is preferred that the contact withthe hydroxycarboxylic acid be made promptly after the contact with themetal chloride. It is also preferred that the earthworm be washed withwater before being brought into contact with the hydroxycarboxylic acid.By removing the metal chloride by washing with water and then bringingthe earthworm info contact with the hydroxycarboxylic acid, a dryearthworm powder having a high enzyme activity can be obtained. When theearthworm is washed with water before the contact with thehydroxycarboxylic acid, the washing of the earthworm with water isperformed preferably within 30 minutes, more preferably within 20minutes, after the initiation of the contact with the metal chloride.The method of washing the earthworm with water is not particularlyrestricted, and a known method can be employed.

If live earthworms are left in contact with hydroxycarboxylic acidpowder for a long time, the earthworms are killed, so that their vitalfunctions are lost and the digested matters in their digestive tractsare no longer excreted. Therefore, it is preferred to dilute thehydroxycarboxylic acid with water as soon as possible, preferably within30 seconds, more preferably within 20 seconds, so as to adjust the pH toa range of 2 to 5.

Since a hydroxycarboxylic acid creates a living environment unpleasantto earthworms, live earthworms, following their self-preservationinstinct, try to improve the living environment through discharge ofbody fluids and excretion. Further, since hydroxycarboxylic acids havedisinfecting properties, they are expected not only to play a role inpromoting excretion of digested matters and the like remaining in thedigestive tract as described above, but also to have an effect ofkilling bacteria adhering to earthworms.

In the above-described method, any crystalline hydroxycarboxylic acidcan be used regardless of the number of its hydroxy groups and carboxylgroups, as long as it assumes a crystalline form under the conditions ofits use. That is, the crystalline hydroxycarboxylic acid may he any ofmonohydroxy monocarboxylic acids, monohydroxy polycarboxylic acids,polyhydroxy monocarboxylic acids and polyhydroxy polycarboxylic acids.

Examples of the hydroxycarboxylic acid used in the present inventioninclude glycolic acid, lactic acid, acetic acid, β-hydroxypropionicacid, α-hydroxy-n-butyric acid, β- hydroxy-n-butyric acid,α-hydroxy-n-valeric acid, β-hydroxy-n-valeric acid, malic acid,α-methylmalic acid, α- hydroxyglutaric acid, β-hydroxyglutaric acid,citric acid, malonic acid and succinic acid. Thereamoag, lactic acid,acetic acid, malic acid, citric acid, malonic acid and succinic acid arepreferred because they can be used in food products and easily obtained.The above-described hydroxycarboxylic acids may be used individually; ortwo or more thereof may be used in combination.

Water constitutes 65% of the tissues of a live earthworm. Although theself-preservation functions of a live earthworm remain effective for acertain time, the death of the live earthworm results in the onset ofenzyme activities; therefore, it is required to carefully control theperiod of placing the live earthworm under an unpleasant livingenvironment. The duration of this period varies depending on theconditions; however, it is usually in a range of 3 to 180 minutes.

It is preferred that the thus hydroxycarboxylic acid-treated liveearthworm be washed with water and then ground into a liquid-form orpaste-form ground product. The washing is preferably performed with purewater. The washing method is not particularly restricted, and a knownwashing method with water can be employed. The total time of thetreatment process before the grinding, that is, the duration of theperiod from the sprinkling of the metal chloride on the live-earthwormto the completion of the removal of the hydroxycarboxylic acid bywashing with water, is preferably not longer than 240 minutes.

The grinding method is not particularly restricted and, for example, thegrinding is usually performed at 1 to 25° C. by using a homogenizer, ablender, a homomixer, a grinder, a high-pressure cell-crushing apparatusor the like. From the standpoint of inhibiting degradation of theearthworm components, it is preferred that the grinding be performed ata low temperature, more preferably at a temperature of 2 to 15° C.

The ground product obtained by grinding the earthworm is placed on astainless-steel tray or the like and subjected to freeze-drying. In thisprocess, a decomposition gas may be generated because the enzymescontained in the living body of the earthworm are inactive in the livingcells but act instantaneously on dead cells. In order to inhibit thegeneration of a decomposition gas, it is preferred that, before beingfreeze-dried, the ground product be momentarily cooled rapidly andfrozen at −18° C. to −35° C. so as to suppress the enzyme actions.

In this manner, for the preparation of earthworm powder withoutimpairing the inherent pharmacological actions of the earthworm, it ispreferred that the ground earthworm be quickly frozen. On the otherhand, an excessively rapid freezing is not preferred because when theground earthworm is frozen in an excessively short period of time, theimpurities existing together with the proteins that are major componentsof an earthworm paste can form spots of unfrozen parts and thus may nothe separated. Therefore, the freezing is performed at a low temperatureof −18° C. to −35° C. over a period of preferably 20 to 240 hours, morepreferably 50 to 170 hours.

For the freeze-drying, it is important to select conditions that allowremoval of water as well as impurities without leaving any impurity.Accordingly, it is preferred that the freeze-drying be performed under apressure of 50 Pa or less while increasing the temperature stepwise in arange of −60° C. to +90° C. over a period of 10 to 60 hours.

As a freeze-drying method, for example, as described above, afterfreezing the ground product at a temperature of −18° C. to 35° C. over aperiod of 20 to 240 hours, the resultant is vacuum freeze-dried over aperiod of 10 to 60 hours while increasing the temperature in severalsteps in a range of −60° C. to +90° C. and reducing the pressure inseveral steps in a range of 25 to 40 Pa, whereby a sterile pale yellowdry earthworm powder can be obtained.

Further, it is also preferred to incorporate the steps of dissolving thethus freeze-dried ground product in water or an aqueous ethanolsolution; and removing or separating insoluble fractions. The step ofremoving or separating insoluble fractions can be performed in the samemanner as described above and comprises precipitation carried out byleaving the resulting solution to stand, centrifugation, filtration andthe like. The step of dissolving the freeze-dried ground product inwater or an aqueous ethanol solution is preferably performed withstirring or shaking. The time required for dissolution of thefreeze-dried ground product in water is preferably 1 to 120 minutes,more preferably 5 to 80 minutes. The ethanol concentration of theaqueous ethanol solution is not particularly restricted; however, it ispreferably 10 to 70% (v/v), more preferably 30 to 60%.

As the Tau protein production accelerator, therapeutic agent, preventiveagent and food composition of the present invention, a supernatantobtained from water or aqueous ethanol solution in which freeze-driedground earthworm is dissolved as described above may be used as it is inthe state of an aqueous solution, or may be used in the form of aconcentrate after evaporating water therefrom. The supernatant may alsobe dried to be used in a powder form, and the powder obtained by dryingthe supernatant may be dissolved in water for use. Further, powderobtained by freeze-drying an earthworm paste can be used as it is,without being dissolved in water or an aqueous ethanol solution.

In the present invention, as a removal method, before the treatment ofplacing live earthworms under an unpleasant environment, that is, beforebringing live earthworms into contact with a metal chloride or ahydroxycarboxylic acid as described above, it is preferred that the liveearthworms be transferred to a flat box such as a bread box and left tostand for 10 to 50 hours in a bright place, followed by removal of dirtadhering to the earthworm skin. The duration of leaving the liveearthworms in a bright place is more preferably 12 to 24 hours. In thisprocess, it is preferred that the amount of the live earthwormscontained in the flat box be such an amount that the earthworms arepiled up to a thickness of 30 to 60 mm, preferably 40 to 50 mm. Careshould be taken so that the flat box contains no foreign matter such assand or mud and, since earthworms are nocturnal and thus become activein their living activities in dark place and this may lead to theirphysical exhaustion, it is preferred to employ a light culture method orthe like during the night so as to keep the flat box under a brightcondition. This treatment makes the live earthworms exhibit theirself-protection instinct and try to maintain their living environment byexcreting the digested matters remaining in their digestive tracts,covering their entire body with the excrements and thereby preventingevaporation of water. Thus, by repeatedly striping off this coveringdirt, namely excrements, by an appropriate means, the digested mattersin the digestive tracts of the earthworms and the dirt adhering to theirskin can be eventually removed.

The dirt adhering to the earthworms' skin can be stripped off by, forexample, covering the live earthworms with a nonwoven fabric andallowing the dirt to adsorb to the fabric. By performing this process ofleaving the earthworms in a bright place and removing the dirt adheringto their skin in combination with the above-described process ofbringing the earthworms into contact with the a metal chloride and/or ahydroxycarboxylic acid, further excretion and removal of toxic matterscontained in the earthworms' body can be expected.

In the present invention, as a method for obtaining a dry earthwormpowder, the following methods are preferred particularly from thestandpoint of the storage stability of the resulting dry powder.

(A-1) A method of producing a dry earthworm powder, the methodcomprising the steps of:

bringing a live earthworm into contact with a chloride of at least onemetal selected from the group consisting of potassium, sodium, magnesiumand calcium; and

subsequently bringing the live earthworm into contact with a powder-formhydroxycarboxylic acid, diluting the resultant with water to adjust thepH to 2 to 5, maintaining the resulting dilution for 3 to 180 minutes,washing the live earthworm with water, grinding the live earthworm andthen freeze-drying the thus obtained ground product.

(A-2) A method of producing a dry earthworm powder, the methodcomprising the steps of:

bringing a live earthworm into contact with a chloride of at least onemetal selected from the group consisting of potassium, sodium, magnesiumand calcium; and

subsequently immersing and maintaining the live earthworm for 3 to 180minutes in an aqueous hydroxycarboxylic acid solution having an adjustedpH of 2 to 5, washing the live earthworm with water, grinding the liveearthworm and then freeze-drying the thus obtained ground product.

(A-3) The method of producing a dry earthworm powder according to theabove-described (A-1) or (A-2), which further comprises the steps of:dissolving the thus freeze-dried ground product into water or an aqueousethanol solution; removing or separating insoluble fractions; and thenfurther freeze-drying the resultant.

Further, after the freeze-drying of the ground product obtained bygrinding the live earthworm, from the standpoint of sterilization of theresulting dried product, the dried product is preferably heat-treated ata temperature of 110° C. or higher but lower than 130° C. When theheating temperature is lower than 110° C., the dried product may not besterilized sufficiently, whereas when the heating temperature is 130° C.or higher, the enzymes contained in the dried earthworm product areinactivated and their activities are thus reduced, which is notpreferred. The heating temperature is more preferably 115° C. to 125° C.The heating method is not particularly restricted, and examples thereofinclude a method of blowing hot air; a method using a heating jacket; amethod of heating the subject on a tray or the like using a heater; anda method using a thermostat incubator. The heating time is preferably 30seconds to 130 minutes, more preferably 30 minutes to 90 minutes, stillmore preferably 60 minutes to 90 minutes. An excessively short heatingtime may result in insufficient sterilization while an excessively longheating time may cause the loss of enzyme activities, neither of whichis preferred. When enzymes contained in a liquid are subjected to theabove-described heat treatment, the activities of the enzymes are lost;therefore, in the present invention, it is preferred to use a dryearthworm powder.

In the present invention, as a method for obtaining a ground product ofan earthworm, the following methods are preferred.

(B-1) A method of producing a ground product of an earthworm, the methodcomprising the steps of:

bringing a live earthworm into contact with a chloride of at least onemetal selected from the group consisting of potassium, sodium, magnesiumand calcium; and

subsequently bringing the live earthworm into contact with a powder-formhydroxycarboxylic acid, diluting the resultant with water to adjust thepH to 2 to 5, maintaining the resulting dilution for 3 to 180 minutes,washing the live earthworm with water and then grinding the liveearthworm.

(B-2) A method of producing a ground product of an earthworm, the methodcomprising the steps of:

bringing a live earthworm into contact with a chloride(s) of a metal(s)selected from the group consisting of potassium, sodium, magnesium andcalcium; and

subsequently immersing and maintaining the live earthworm for 3 to 180minutes in an aqueous hydroxycarboxylic acid solution having an adjustedpH of 2 to 5, washing the live earthworm with water and then grindingthe live earthworm.

In the present invention, as a method for obtaining an earthwormextract, the following methods are preferred.

(C-1) A method of producing an earthworm extract, the method comprisingthe steps of:

bringing a live earthworm into contact with a chloride of at least onemetal selected from the group consisting of potassium, sodium,magnesium, and calcium; and

subsequently bringing the live earthworm into contact with a powder-formhydroxycarboxylic acid, diluting the resultant wife water to adjust thepH to 2 to 5, maintaining the resulting dilution, for 3 to 180 minutes,washing the live earthworm with water, grinding the live earthworm,freeze-drying the resulting ground product, dissolving the thusfreeze-dried product in water or an aqueous ethanol solution, and thenremoving or separating insoluble fractions.

(C-2) A method of producing an earthworm extract, the method comprisingthe steps of:

bringing a live earthworm into contact with a chloride(s) of a metal( s)selected from the group consisting of potassium, sodium, magnesium andcalcium; and

subsequently immersing and maintaining the live earthworm for 3 to 180minutes in an aqueous hydroxycarboxylic acid solution having an adjustedpH of 2 to 5, washing the live earthworm with water, grinding the liveearthworm, freeze-drying the resulting ground product, dissolving thethus freeze-dried product in water or an aqueous ethanol solution, andthen removing or separating insoluble fractions.

EXAMPLES

The present invention will now he described in more detail by way ofexamples thereof. The present invention, however, is not restricted bythe following examples by any means. It is noted here that, unlessotherwise specified, “%” used below is all based on mass.

[Preparation of Dry Earthworm Powder]

After leaving 30 kg of live red earthworms (Lumbricus rubellus) to standin a bright place for 24 hours and stripping off dirt adhering to theirskin, the live red earthworms were spread at a thickness of about 5 cmon a flat dish, and 250 g of sodium chloride was uniformly sprinkledthereon. The earthworms were washed with water 20 minutes thereafter.Then, 15 seconds after sprinkling 250 g of citric acid on the earthwormsin the same manner, 30 L of pure water was added thereto for dilution.In this process, the pH of the resulting solution was 2.25 immediatelyafter the addition of water and 2.74 after the completion of thedilution. When sprinkled with the citric acid powder, the earthwormsexcreted yellow body fluid at once. After the dilution with water, theearthworms were maintained In this state for 20 minutes. Next, the liveearthworms were taken out of the resulting dirty aqueous citric acidsolution, washed with water and subsequently ground at 10° C. using ahomogenizer to prepare an earthworm paste. Then, after subjecting thisearthworm paste to vacuum degassing so as to remove the gases containedtherein, the earthworm paste was transferred to a stainless-steel traywhere the earthworm paste was instantaneously and rapidly cooled to −35°C. and maintained at this temperature for 50 hours to be slowly frozen.The thus frozen earthworm paste was maintained at −35° C. under apressure of 0 Pa for 2 hours. Thereafter, the earthworm paste was heatedand dried at 25° C. under a pressure of 40 Pa for 10 hours, at 40° C.under a pressure of 35 Pa for 14 hours and then at 65° C. under apressure of 35 Pa for 12 hours and lastly; the resultant was maintainedat a temperature of 80° C. and a pressure of 25 Pa for 6 hours, therebyvacuum freeze-drying the earthworm paste. By this treatment, apale-yellow dry earthworm powder having a water content of 8% by masswas obtained.

The thus obtained dry earthworm powder was heat-treated using a heatingapparatus RM-50D (manufactured by Okawara MFG. CO., Ltd.). As for theheating conditions, the dry earthworm powder was heated to 12° over aperiod of 90 minutes, maintained at 120° C. for 20 minutes and thencooled to 40° C. over a period of 240 minutes. Thereafter, the dryearthworm powder was taken out.

The thus heat-treated dry earthworm powder was dissolved in 50% aqueousethanol solution such that a ratio, ethanol:freeze-dried powder, of 20:1(v/w) was attained, and the resulting solution was shaken for 1 hour at1,500 rpm under room temperature (25° C). Then, the solution wascentrifuged for 15 minutes at 4° C. and 10,000×g, and the resultingsupernatant was separated and vacuum-concentrated at 75° C. for 15minutes. This supernatant was transferred to a stainless-steel traywhere the supernatant was instantaneously and rapidly cooled to −35° C.and maintained at this temperature for 50 hours to be slowly frozen. Thethus frozen earthworm paste was maintained at −35° C. under a pressureof 0 Pa for 2 hours, Thereafter, the earthworm paste was heated anddried at 25° C. under a pressure of 40 Pa for 10 hours, at 40° C. undera pressure of 35 Pa for 14 hours and then at 65° C. under a pressure of35 Pa for 12 hours and lastly, the resultant was maintained at atemperature of 80° C. and a pressure of 25 Pa for 6 hours, therebyvacuum freeze-drying the earthworm paste to obtain a dry earthwormpowder A-1.

[Rat Hippocampal Neuron Culture]

Nerve cells (neurons) isolated from the hippocampal region of a ratembryo at day 18 of gestation were cultured at 37° C. for 7 days.

[Quantification of Tau Protein and Phosphorylated Tau Protein]

The thus cultured rat hippocampal neurons were cultured at 37° C. in 1ml of freeze-dried earthworm powder-dissolved culture media eachprepared by dissolving the above-obtained dry earth worm powder A-1 in aculture medium, Neural Media 5 (NM5), at a concentration of 0, 1, 10,100 or 1,000 ng/mL (cell number: 1×10⁶/12 well dish). The formulation ofthe culture medium NM5 is as follows:

230 mL of Neurobasal (Gibco 21103-049)

12.5 mL of horse serum (Sigma H1270)

2.5 mL of penicillin/streptomycin (Gibco 15140122)

5 mL Glutamax 1 (Gibco 35050-061)

2% B27 supplement (Gibco 17504044)

Subsequently, after an arbitrary time period, the rat hippocampalneurons were recovered, washed twice with PBS(−) solution and thenadjusted with a sample buffer, and the amount of Tau protein and that ofphosphorylated Tau protein were evaluated by Western blotting. Theprotein amount and phosphorylation amount were determined using an imageanalysis software, ImageJ64, based on the results of Western blotting.The protein amount and phosphorylation amount were also evaluated in thesame manner for Akt and GSK-3β molecules positioned in the signalingupstream of Tau protein.

In the rat hippocampal neurons that were cultured in the dry earthwormpowder-dissolved culture media, no significant increase in the proteinamount was found for AKt and GSK-3/β (FIGS. 1, 2B and 2C); however, itwas found that the amount of Tau protein was increased and that theprotein was produced in more than double the amount as compared to thecontrol “−”; in a culture medium NM5) (FIGS. 1 and 2A). In addition,with regard to the increase in the amount of Tau protein attained by theuse of the dry earthworm powder-dissolved culture media, the maximumincrease was found at a dry earthworm powder concentration of 100 ng/mland a culture time of 48 hours (FIGS. 3 and 4).

[Intracellular Localization of Tau Protein and Phosphorylated TauProtein]

The above-cultured rat hippocampal neurons were cultured at 37° C. in 2ml of a solution prepared by dissolving the above-obtained dry earthwormpowder A-1 in a culture medium NM5 at a concentration of 100 ng/mL (cellnumber: 2×10⁵/6 well dish (with a cover glass on the bottom)).

Then, after 48 hours, the rat hippocampal neurons were recovered andfixed with 4% paraformaldehyde, and intracellular localization wasexamined for Tau protein and phosphorylated Tau protein byimmunostaining.

In the control (“None”; cultured in a culture medium NM5), even theneurites were clearly observed with phosphorylation of Tau protein;however, in the hippocampal neurons cultured in the dry earthwormpowder-dissolved culture medium, no clear image of phosphorylation wasconfirmed at the neurites (FIG. 5).

From the above results, it is seen that dry earthworm powder not onlysignificantly increases the amount of Tau protein but also reduces theamount of phosphorylation especially at the neurites.

The invention claimed is:
 1. A method of producing a Tau proteinproduction accelerator, comprising the steps of: bringing a liveearthworm into contact with a chloride of at least one metal selectedfrom the group consisting of potassium, sodium, magnesium and calcium;subsequently bringing the live earthworm into contact with a powder-formhydroxycarboxylic acid, diluting the resultant with water to adjust thepH to 2 to 5, maintaining the resulting dilution for 3 to 180 minutes,washing the live earthworm with water, grinding the live earthworm andthen freeze-drying the thus obtained ground product; and subsequentlyheat-treating the thus obtained freeze-dried product at a temperature of110° C. or higher but lower than 130° C.
 2. A method of producing a Tauprotein production accelerator, comprising the steps of: bringing a liveearthworm into contact with a chloride of at least one metal selectedfrom the group consisting of potassium, sodium, magnesium and calcium;subsequently immersing and maintaining the live earthworm for 3 to 180minutes in an aqueous hydroxycarboxylic acid solution having an adjustedpH of 2 to 5, washing the live earthworm with water, grinding the liveearthworm and then freeze-drying the thus obtained ground product; andsubsequently heat-treating the thus obtained freeze-dried product at atemperature of 110° C. or higher but lower than 130° C.
 3. The methodaccording to claim 1, wherein the step of heat-treating is performed for30 seconds to 130 minutes.
 4. The method according to claim 2, whereinthe step of heat-treating is performed for 30 seconds to 130 minutes. 5.The method according to claim 1, further comprising the steps ofdissolving the thus heat-treated product in water or an aqueous ethanolsolution; and then removing or separating insoluble fractions.
 6. Themethod according to claim 2, further comprising the steps of dissolvingthe thus heat-treated product in water or an aqueous ethanol solution;and then removing or separating insoluble fractions.
 7. The methodaccording to claim 1, further comprising the steps of dissolving thethus heat-treated product in water or an aqueous ethanol solution;removing or separating insoluble fractions; and then furtherfreeze-drying the resultant.
 8. The method according to claim 2, furthercomprising the steps of dissolving the thus heat-treated product inwater or an aqueous ethanol solution; removing or separating insolublefractions; and then further freeze-drying the resultant.